Distinct regulatory networks control the development of macrophages of different origins in zebrafish.

Macrophages are key components of the innate immune system and play pivotal roles in immune response, organ development, and tissue homeostasis. Studies in mice and zebrafish have shown that tissue-resident macrophages derived from different hematopoietic origins manifest distinct developmental kinetics and colonization potential, yet the genetic programs controlling the development of macrophages of different origins remain incompletely defined. In this study, we use zebrafish, where tissue-resident macrophages arise from the rostral blood island (RBI) and ventral wall of dorsal aorta (VDA), the zebrafish hematopoietic tissue equivalents to the mouse yolk sac and aorta-gonad-mesonephros for myelopoiesis, to address this issue. We show that RBI- and VDA-born macrophages are orchestrated by distinctive regulatory networks formed by the E-twenty-six (Ets) transcription factors Pu.1 and Spi-b, the zebrafish ortholog of mouse spleen focus forming virus proviral integration oncogene B (SPI-B), and the helix-turn-helix DNA-binding domain containing protein Irf8. Epistatic studies document that during RBI macrophage development, Pu.1 acts upstream of Spi-b, which, upon induction by Pu.1, partially compensates the function of Pu.1. In contrast, Pu.1 and Spi-b act in parallel and cooperatively to regulate the development of VDA-derived macrophages. Interestingly, these two distinct regulatory networks orchestrate the RBI- and VDA-born macrophage development largely by regulating a common downstream gene, Irf8. Our study indicates that macrophages derived from different origins are governed by distinct genetic networks formed by the same repertoire of myeloid-specific transcription factors.

The murine placenta contains hematopoietic stem cells within the vascular labyrinth region.

In the midgestation murine embryo, several major vascular tissues contain hematopoietic stem cell (HSC) activity. These include the aorta-gonad-mesonephros region (AGM), yolk sac, and fetal liver. Recently, the placenta was demonstrated to harbor hematopoietic progenitors, but it was not examined for HSC activity. We demonstrate here that the placenta also harbors adult-repopulating HSCs. Placental HSCs begin to be detected at embryonic day (E) 11, and HSC numbers increase dramatically between E11 and E12, exceeding the numbers in the circulating embryonic blood. Furthermore, all placental HSC activity is restricted to the GFP+ fraction of cells in Ly-6A (Sca-1) GFP transgenic embryos. Cells coexpressing GFP and endothelial markers CD34 and CD31 are found in the embryonic vasculature of the placental labyrinth. Moreover, placental cell expression of other HSC markers and transcription factors suggests that HSC emergence may occur in the placenta, as has been proposed for other embryonic hematopoietic sites.

The first 3 days of B-cell development in the mouse embryo.

B-lineage-committed cells are believed to arise in the liver of mouse embryos at 14 days after coitus (dpc). However, pre-B-specific gene transcripts and DJH gene rearrangements have been detected in earlier, midgestation embryos. We describe here a population of c-kit(+)AA4.1(+)CD19(+)Pax5(+) cells present in the aorta-gonad-mesonephros (AGM) area and in the livers of 11-dpc mouse embryos. In contrast to multipotent c-kit(+)AA4.1(+)CD19(-) hematopoietic stem cells (HSCs), these c-kit(+)AA4.1(+)CD19(+) progenitors differentiated only to B-lineage cells in vitro. We propose that mouse embryonic B lymphopoiesis starts earlier than previously thought, at 10 to 11 dpc, both in liver and extra-liver hematopoietic sites. The B-cell differentiation program is not delayed with respect to the emerging lymphohematopoiesis events in the midgestation mouse embryo (8-9 dpc).

Immature multipotent hemopoietic progenitors lacking long-term bone marrow-reconstituting activity in the aorta-gonad-mesonephros region of murine day 10 fetuses.

Previous studies indicated that multipotent progenitors exist in early fetuses that do not contain long-term reconstituting (LTR) activity. However, it remained unclear whether these multipotent progenitors are committed to the hemopoietic lineage or are immature mesodermal cells or hemangioblasts. In this study, we have succeeded in enriching the multipotent progenitors that are capable of generating myeloid, T, and B cells in the LFA-1(-) subpopulation of TER-119(-)c-kit(+)CD45(+) cells from the aorta-gonad-mesonephros (AGM) region of day 10 fetuses. We found that these day 10 AGM LFA-1(-) cells do not show the LTR activity, whereas day 11 AGM LFA-1(-) cells do have such an activity. These results strongly suggest that multipotent progenitors lacking LTR activity emerge as CD45(+) hemopoietic progenitor cells in the AGM region on the 10th day of gestation, and such p-Multi mature into hemopoietic stem cells by acquiring LTR activity.

Emergence of T, B, and myeloid lineage-committed as well as multipotent hemopoietic progenitors in the aorta-gonad-mesonephros region of day 10 fetuses of the mouse.

We investigated the developmental potential of hemopoietic progenitors in the aorta-gonad-mesonephros (AGM) region, where the definitive type hemopoietic progenitors have been shown to emerge before the fetal liver develops. By using an assay system that is able to determine the developmental potential of individual progenitors toward the T, B, and myeloid lineages, we show that not only multipotent progenitors but also progenitors committed to the T, B, or myeloid lineage already exist in this region of day 10 fetuses. Bipotent progenitors generating myeloid and T cells or those generating myeloid and B cells were also detected, suggesting that the commitment to T and B cell lineages is in progress in the AGM region. The numbers of these progenitors, however, were only 1/200-1/1000 of those in fetal liver of day 12 fetuses. Such small numbers of progenitors suggest that hemopoiesis has just started in the AGM region of day 10 fetuses. Although most of T cell lineage-committed progenitors in the AGM region generated only a small number of immature T cells, some were able to generate a large number of mature T cells. The detection of various types of lineage-committed progenitors strongly suggests that the AGM region is not only the site of stem cell emergence, but also the site of hemopoiesis, including lineage commitment. The T cell progenitors found in the AGM region may represent the first immigrants to the thymus anlage.

Immunoglobulin-containing cells in chick embryo urogenital tissues: a new site for early B lineage cells in endothermic vertebrates.

We have employed histological and immunofluorescent staining procedures in order to characterize the distribution of mu + B lineage cells in tissue sections prepared from developing chicken embryo urogenital tissues (UGTs) between 14 and 21 days of incubation. B lineage cells were observed in tissue sections prepared from developing UGTs, especially the mesonephros and its associated tissue, throughout the sample period. The highest densities of mu + B lineage cells were observed in tissue sections prepared from 18 day embryos. The mu + UGT cells were distributed singly and in clusters in subcapsular regions and within the peritubular interstitium of the mesonephros. These observations (1) are consistent with those which suggest nonbursal site(s) for origin of cells in B lineage, (2) may help account for the varying effects of embryonic caudectomy performed between the second and third days of incubation and surgical bursectomy performed close to hatching, (3) may help provide new insights on the effects of sex hormones on B cell development, and (4) suggest fundamental ontogenetic and phylogenetic similarities between developing vertebrate immune systems.

Demonstration of human kidney differentiation antigens with monoclonal antibodies.

Six human differentiation antigens (EE24.6, EG9.11, EG14.1, EI16.1, EK8.1, EK17.1) have been defined using monoclonal antibodies obtained from mice immunized with embryonic kidney cells. Their histologic distribution was determined on frozen sections of embryonic, fetal, and adult human kidneys by immunofluorescence assay. EE24.6, an ureteral bud marker, was detected only on the germ layer of mature kidney urothelium. EG9.11 and EG14.1 were detected on the S-shaped bodies and also on the adult proximal convoluted tubule for the former and the glomerular basement membrane for the latter. EI16.1, a marker of condensed mesenchyme, was detected only on epithelial cells of adult proximal convoluted tubule. EK8.1 was found in the mesangium, connective tissue, and with particularly dense labeling in the basement membranes. This labeling pattern was present throughout renal organogenesis. EK17.1 recognized both cell and plasma human fibronectins. Staining for all antibodies was nearly identical in mesonephros and metanephros. These results demonstate that some antigens follow their embryonic destiny. They indicate an antigenic similarity between the mesonephros and the metanephros and, therefore, a very early appearance of these antigens. During differentiation, these antigens concentrate on more defined structures, and staining became increased with an increased degree of differentiation.

Analysis of structural properties and cellular distribution of avian Ia antigen by using monoclonal antibody to monomorphic determinants.

In this report, we describe the analysis of Ia-like antigens in the chicken by using a monoclonal antibody (CIa-1) reactive with monomorphic determinants of the Ia-like (B-L) antigens. This antibody reacts with determinants on B cells in all avian species tested, but does not detect antigens on lymphocytes of representative mammals, reptiles, and amphibians. In addition to B cells, this antibody defines a subpopulation of the monocyte-macrophage series and reacts with mitogen-activated T cells. Immunochemical analysis indicates that the CIa-1 reactive antigen is a 65,000-dalton glycoprotein consisting of an alpha-chain of 32,000 daltons noncovalently bound to a beta-chain of 27,000 daltons. Under nonreducing conditions, the beta-chain migrates with slightly faster mobility. Two-dimensional gel analysis indicates that the beta-chain is the more heterogeneous of the two chains. Thus, the antigen detected by CIa-1 antibody is similar in cell distribution and structure to the murine Ia antigens and human DR antigens. During in ovo development, Ia+Ig- cells were not found in the yolk sac but were detected in the spleen, mesonephros, and bursa of 9-day embryos. Two populations of Ia+Ig- cells were identified in the bursa: 40 to 60% of the bursacytes, mostly larger cells, exhibited brighter immunofluorescence reactivity than the smaller bursacytes.

Localization of immune complexes and heat-aggregated immunoglobulin in the carp Cyprinus carpio L.

In carp Cyprinus carpio L. preimmunized with a protein antigen, human gamma globulin (HGG), rapid localization of antigen occurred in the splenic ellipsoids within 1 day of a secondary immunization given 8 weeks later. Furthermore, after injection of immune complexes or heat-aggregated HGG, antigen was trapped more quickly in the pronephros than when antigen was injected alone. These results support the conclusion that in fish, as in homoiotherms, this form of antigen trapping is antibody-dependent.

[Organ specific embryonic antigens during the differentiation of the liver and kidney in chickens]. / Recherche d'antigènes embryonnaires spécifiques d'organe au cours de la différenciation du foie et du rein chez le Poulet

Among the cellular antigens, demonstrated by their reaction with Rabbit anti-embryonic Chick liver serum, two are present in the supernatant fraction obtained after ultracentrifugation of liver extracts from 6-day-old Chick embryos, and in the sedimentable fraction from the 9th day onward. A third antigen, detectable from the 9th day of incubation, is found only in the sedimentable fraction. The three antigens exist, but at much lower concentrations, in adult Chick kidney. Rabbit anti-embryonic Chick mesonephros serum permits the demonstration of at least one kidney-specific antigen. The latter, localized at the apical part of the cells of the proximal secretory tubules, is present in mesonephros and embryonic or adult metanephros. Although suggested by several observations, the presence of antibodies reacting with mesonephros-specific antigens could not be definitely established.

Nueva Aportacion al estudio de la Reaccion de Wolff en nuestro medio

Realizado el estudio, del presente trabajo, podemos sacar las siguientes conclusiones, 1.- En los casos en que la reacciòn fuè negativa no encontramos gramos de hemozoina en ninguna de las placas respectivas, 2.- Hipotèticamente, la reacciòn es producida por la presipitaciòn de la hemozoina por el reactivo, 3.- El ìndice de positividad de la reacciòn es de 97,2 porciento, 4.-En los individuos que hayan enfermado paludismo y recibido un tratamiento intenso, el resultado de la reacciòn es siempre negativo, 5.- Por su facìl realizaciòn y pequeño costo econòmico, se la recomienda como una reaccion de rutina